A research program will be undertaken to study the structure, function and regulation of the adaptor protein, cCrk-ll, which plays an important role in cellular signaling events such as proliferation, differentiation, cell adhesion and cytoskeletal reorganization. There is mounting evidence, including our own preliminary data, for significant structural and functional interplay between the individual modular domains in this proto-oncogene. These intramolecular interactions appear to be important for the normal regulation of this molecule. Indeed, mutant forms of the Crk protein (as well as the viral oncogene, v-Crk) lacking various autoregulatory domains are known to transform cells. To better understand the molecular mechanisms of autoregulation, we will use protein engineering approaches, newly developed in our laboratory, to study the effect of native context on the stability, folding kinetics, backbone dynamics and function of the three component Src Homology Domains in the context of full-length c-Crk-fl. Key to this research program is the protein semi-synthesis technology, expressed protein ligation, which allows biochemical and biophysical probes to be site-specifically introduced into large proteins. Our studies will also provide much needed structural and functional information on the poorly understood C-terminal third of c-Crk-ll molecule, a region that we hypothesize regulates the function of the protein through intramolecular interactions. Lastly, we propose to develop photochemical strategies to control the regulatory state of c-Crk-ll with light. Specifically, we will prepare semi-synthetic c-Crk-H analogs containing photocaged phosphorylated amino acid derivatives at position Tyr22 1. Proof ofprinciple studies will then be performed to explore whether we can photochemically trigger phosphorylation of Y 21 in c-Crk-II, thereby controlling the regulatory state of the protein. Ultimately, this methodology will allow the controlled and synchronized release of active phospho-protein into cells with second-range kinetic resolution. The precise temporal and spatial control of delivery will provide information on the localization, trafficking, and metabolism of the target proteins. In summary, many biomedically important proteins, including a large number of oncogenes, contain multiple protein modules. There is pressing need to develop approaches that provide a way of extracting detailed structural and functional information on a specific domain within the context of a large multi-domain protein. The approaches developed in this proposal will be generally applicable to these types of structural and biochemical problems.